Viability Dyes
INTRODUCTION
When analysing cells by flow cytometry, it is important that the data being acquired and analysed is
based only on the cells that we are interested in; therefore for most experiments the exclusion of dead
or dying cells is important. A simple method of distinguishing dead or dying cells is to add a brightly
fluorescent DNA binding dye that can enter the cell without necessary treatment.
There are many dyes that can be used to distinguish healthy cells from dying cells and the use of these
dyes is principally the same. Dyes such as Propidium Iodide (PI), 7 Amino-Actinomycin D (7AAD),
4’, 6 – diamidine – 2 – phenylindole (DAPI) and TO-PRO-3 can be used on unfixed, live cells.
In addition there are also fixable viability dyes used to identify dead cells post-fixation and postpermeabilisation, which fluoresce in various colours. These dyes, which bind to proteins, are
especially useful as cells can then be processed and stained for intracellular markers and still allow the
exclusion of non-viable cells.
REAGENTS
Dyes for Unfixed Cells
Propidium Iodide (PI) (Sigma; Cat No: P4170)
Stock Solution 50µg/ml in PBS (Phosphate Buffered Saline)
Store at 4°C
7-Amino-Actinomycin D (7AAD) (Sigma; Cat No: A9400)
Make up 1mg/ml in DMSO (Dimethylsulphoxide) initially, then prepare Stock Solution 40µg/ml in
PBS
Store at 4°C
TO-PRO-3 (Molecular Probes; Cat No T3605)
Stock Solution 1mM in DMSO, make up 1µM from this in PBS
Store at 4°C
4’, 6 – Diamidino – 2 – phenylindole (DAPI) (Sigma; Cat No: D9542)
Stock Solution 200µg/ml in Distilled H2O
Store at 4°C
DRAQ7 (Biostatus; Cat No: DR71000)
Stock Solution 0.3mM
Store at 4oC
Please note that these dyes are mutagenic and carcinogenic; please exercise caution when handling
these dyes. See FACS Lab PRA04.
Dyes for Fixed Cells
Live/ Dead® Fixable Blue Dead Cell Stain Kit (Invitrogen – Molecular Probes Cat No: L23105)
Live/ Dead® Fixable Near IR Dead Cell Stain Kit (Invitrogen – Molecular Probes Cat No: L10119)
Live/ Dead® Fixable Aqua Dead Cell Stain Kit (Invitrogen – Molecular Probes Cat No: L34957)
Live/ Dead® Fixable Red Dead Cell Stain Kit (Invitrogen – Molecular Probes Cat No: L23102)
In each kit, a vial of the fluorescent reactive dye (Component A) and a vial of 500µl of anhydrous
DMSO (Component B) is provided.
Stock Solutions add 50µl of the DMSO to the dye component, once the DMSO has been added to the
dye, ensure that it is protected from light. To ensure that the dye remains stable, aliquot the prepared
solution into smaller volumes so not to freeze/thaw the entire dye solution.
Store at -20°C, once the vial is thawed do not refreeze.
Zombie Aqua Fixable Dye (Biolegend – Cat No: 77143)
Zombie Yellow Fixable Dye (Biolegend – Cat No: 77168)
Zombie Near Infra Red Fixable Dye (Biolegend – Cat No: 77184)
eFluor780 Fixable Dye (eBioscience Cat No: 65-0865-14)
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Other Fixable Dyes (not tested here)
Ghost Dye Violet 450 (Tonbo Biosciences – Cat no: 13-0863-T100)
Ghost Dye Violet 510 (Tonbo Biosciences – Cat No: 13-0870-T100)
Ghost Dye Red 780 (Tonbo Biosciences – Cat No: 13-0865-T100)
METHOD
UNFIXED CELLS
The volumes added are sufficient to stain approximately 1 x 106 cells in 1 ml of solution.
1.
Add 50µl per ml of 50µg/ml PI .
2.
Add 50µl per ml of 40µg/ml 7AAD.
3.
Add 50µl per ml of 1µM TO-PRO-3.
4.
Add 5µl per ml of 200µg/ml DAPI.
5.
Add 10ul per ml to give final concentration of 3uM DRAQ7. Incubate at RT, in the dark for
10 minutes.
6.
Samples 1 – 4 can be run straight after addition of the dye; for large sample numbers it may be
better to add in batches. Unfixed cells should be kept on ice prior to reading.
FIXED CELLS
Buffers appropriate for cell staining include phosphate-buffered saline (PBS), Hank’s Balanced
Salt Solution (HBSS), and Dulbecco’s PBS. When using an amino-reactive dye, Tris buffers and
solutions containing sodium azide or extraneous protein should NOT be used for cell
resuspension and washing.
1.
After completing your extra-cellular staining, pellet your cells ensuring that they contain at
least 1 x 106 cells.
2.
Wash cells once with PBS, pellet and discard supernatant.
3.
Re-suspend cells in 1ml PBS.
4.
Add 1µl of the reconstituted fluorescent dye to the 1ml cell suspension and mix well.
NB: For the Biolegend dyes add 1ul to 100ul of cells instead.
5.
Incubate at room temperature or on ice for 30 minutes in the dark.
6.
Wash cells once with 1ml of PBS, pellet and discard supernatant.
7.
Fix cells in a final concentration of 2-4% PFA (Paraformaldehyde) or ethanol. Store at 4°C for
at least 30 mins. Results show cells can be stored fixed for up to 72 hours before analysis (see
link to pdf in the Expected Results section below).
8.
Wash and re-suspend in PBS. Cells can be stored at 4°C at this point.
After this the cells are ready to be run on the cytometer or be processed for intracellular staining.
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CYTOMETER SET UP
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EXPECTED RESULTS
Unfixed cells stained with DNA dyes
TO-PRO-3 FL4-H 661/16BP
104
4
105
10
103
2
102
103
101
102
103
104
DAPI: 440/40-UV-A
7AAD: 660/20 yellow-A
103
104
Propidium Iodide FL3-H 670LP
10
105
101
102
0
0
0
50K
(A) 7AAD
100K
150K
FSC-A
200K
0
250K
85.3
81.8
100
87.6
200
400
600
FSC-H
800
0
1000
(B) TO-PRO-3
50K
100K
150K
FSC-A
200K
(C) DAPI
84.1
100
250K
0
200
400
600
FSC-H
50K
100K
150K
FSC-A
800
1000
(D) PI
Cells fixed for 24 hours with 4% Paraformaldehyde
10 3
10 4
10 3
10 5
10 4
10 4
10
3
2
10 2
0
0
10
10 2
10 5
0
780/60-RED-A-A
4
525/50-VIOLET-E-A
10
10 5
15.2
586/15-VIOLET-C-A
780/60-RED-A-A
10 5
0
50K
100K
150K
FSC-A
(A) eFluor780
200K
250K
0
50K
100K
150K
FSC-A
(B) Zombie Yellow
200K
3
10 2
0
83.5
86.3
80
10
0
250K
50K
100K
150K
FSC-A
(C) Zombie Aqua
200K
250K
82.9
0
200K
250K
(D) Invitrogen NIR
TROUBLESHOOTING and TIPS
1.
Cells stained with the LIVE/DEAD fixable dyes are better prepared when washed in
Phosphate Buffered Saline (PBS), it is important to use a buffer that does not contain any
extraneous protein that the amine reactive dye can bind to.
2.
When staining cells with LIVE/DEAD fixable dye best results are achieved at room
temperature in the dark, however depending on the cell type and nature of your cells you may
want to keep them on ice whilst staining.
3.
The intensity of the 7AAD dye degrades gradually over a few months, this will be noticeable
as you compare cells stained with 7AAD when it was freshly prepared and after several
months.
4.
The LIVE/DEAD Fixable Dyes can be excited with different lasers and can be detected in
many channels from the blue, yellow, violet and red detectors. Please note that when using
these dyes compensation will be required between fluorophores. This is especially the case if
the cells are left fixed for a period of time.
REFERENCES and RESOURCES
Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry –
Perfetto, S.P., Chattopadhyay, P.K., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R.A., Roederer, M.
Probes.invitrogen.com/products/flowcytometry
http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html
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